Over the last thirty years, the immune system has been studied with better and better laboratory tools. Still, most knowledge of the immune response concerns antibody formation. This is understandable given that antibodies, including specific antibodies, are easily detectable in quantity in the serum of immunized individuals. Antibodies are products of B-lymphocytes (“B cells” or “B-cells”). Antibody production by individual B cells (as well as cells fused with B cells such as hybridomas) is also readily achieved in vitro using a variety of tests, including the ELISA spot assay (also called “ELISPOT” for “enzyme-linked immunospot”). See Segwick, J. D. and Holt, P. G., “A solid-Phase Immunoenzymatic Technique for the Enumeration of Specific Antibody-Secreting Cells,” J. Immunol. Methods 57:301-309 (1983). See also Mazer, B. D. et al., “An ELISA Spot Assay for Quantitation of Human Immunoglobulin Secreting Cells,” J. Allergy Clin. Immunol. 88:235-243 (1991). Many human diseases involve pathological immune reactions. These include autoimmune disorders, such as systemic lupus erythematosis, Wegner's syndrome, and multiple sclerosis, by way of example only. It can be challenging to treat individuals with such disorders, because treatment of immune-mediated disorders generally includes removal of the patient from the immunogen or antigen responsible for the immune reaction. Unfortunately, in autoimmune diseases, it is very difficult, if not impossible to remove the antigenic stimulus from the patient because the antigen is the patient's own.
In a conventional B cell ELISA spot assay, standard, commercially-available plates are coated overnight with antigen or animal antibody. In the case where antibody is used, it is typically an “anti-antibody” (e.g., goat antibody reactive with human IgG, IgE, IgM, etc.). After blocking overnight, B cells are introduced in the wells. Following a sufficient culture period, the wells are washed free of the cells and an antibody-enzyme conjugate is added. The plates are then developed using substrate for the enzyme of the conjugate. Spots are counted using a microscope. The lowest amount of detectable antibody is typically in the range of 10 to 50 picograms. See e.g., Renz, H. et al., “Enhancement of IgE Production by Anti-CD40 Antibody in Atopic Dermatitis,” J. Allergy Clin. Immunol. 93:658 668 (1994).
In contrast to the antibody response, the response of T-lymphocytes (“T-cells” or T Cells”) to antigen (including the antigen of pathogens) can not be easily monitored due to the fact that antigen reactive T cells occur in low frequencies and the fact that their secretory products are not typically stable (i.e. have a short half-life). Indeed, even in hyperimmunized individuals, antigen reactive T cells constitute 1 in 10,000 cells or less in the peripheral T cell pool, for example, the T cells in circulating blood. Thus, T cells usually act beyond the detection limits of conventional assay systems (such as proliferation assays).
As a consequence of this, there are few sensitive, reliable and rapid techniques at present available that would reliably measure whether a patient has generated a T cell response to a particular pathogen, such as HIV. There is no reliable assay that can detect whether a T cell response to HIV proteins has been generated, what proteins of the virus are primarily targeted, and which determinants within that protein are immunodominant. There is also no reliable method available for testing the magnitude of the anti-viral T cell response (clonal sizes) and its quality (e.g. whether the response is pro- or anti-inflammatory).
Heterogeneity of T cells, their products and the mode of function provide great challenges (particularly as compared to B cells). With respect to mode of function, T cells can act in different subpopulations that utilize strikingly different effector functions. T cell responses can be pro-inflammatory T helper 1 type, Th1, characterized by the secretion of interferon gamma (IFNγ) and interleukin 2 (IL-2). Th1 cells are critical for the cellular defense and provide little help for antibody secretion. (Strong Th1 responses are usually associated with poor antibody production, which highlights the importance of directly measuring the T cell response instead of relying on antibody measurements.) The other class of T cell responses is antiinflammatory, mediated by Th2 cells that produce IL-4, 5, 10, but no IL-2 or IFNγ. Th2 cells are the helper cells for antibody production. CD4+ and CD8+ cells both occur in these subpopulations: Th1/Th2:CD4, TC1/TC2:CD8.
Importantly, for each type of infection there is an “appropriate” (and different) type of T cell response (e.g., Th1 vs. Th2, CD4+ vs. CD8+) that clears the infectious agent but does not cause excessive tissue destruction to the host. It is detrimental to the host if an “inappropriate” type of T cell response is engaged (Th1 instead of Th2 or vice versa). Thus, there is a strong need for assessing the host's T cell immunity to the virus to understand the host-virus interplay and to design vaccines. An ideal assay should permit monitoring all of the critical features of the T cell response: first, the existence of a response, i.e., that effector cells have been generated, second the nature of the effector cells as Th1 or Th2 type cells, and finally the magnitude of the response.
Some attempts have been made to apply the B-cell ELISA spot technology to T cells. However, the conventional cytokine ELISA spot assay has not been a more sensitive tool than alternative assays (e.g. proliferation assays), displaying high background and generally a weak signal. The conventional ELISA spot assay for 1 T cells involves plates containing nitrocellulose membranes that are pre-coated with a capture antibody specific for the cytokine to be detected. See e.g., Taguchi, T. et al., “Detection of Individual Mouse Splenic T Cells Producing IFNγ and IL-5 Using the Enzyme-Linked Immunospot (ELISPOT) Assay,” J. Immunol. Methods 128:65-73 (1990). See also Fujihashi, F. et al., “Cytokine-Specific ELISPOT Assay,” J. Immunol. Methods 160:181-189 (1993). See also Miyahira, Y. et al., “Quantification of Antigen Specific CD8+ T Cells Using an ELISPOT Assay,” J. Immunol. Methods 181:45-54 (1995). T cells are plated with the test antigen and start to secrete the type of cytokine they are programmed to produce. As the cytokine is released, it is captured around the secreting cells by the plate bound antibody. After 24 h the cell culture is terminated, cells are removed and the plate-bound cytokine is visualized by a second antibody and an enzymatic color reaction.
Ideally, each product-producing cell will be represented as an ELISA spot. However, with conventional assays, sensitivity does not exceed cytokine measurements in the supernatant by ELISA (cytokine measurements in culture supernatants provide a positive result only if more than 1000 cells are present per well). The quantification of the data is also problematic because of background problems and the subjective, visual evaluation. Moreover, because enzymatic reactions are sensitive to time, reagent quality and reaction conditions, there can be variations in results obtained using conventional ELISA or ELISPOT methods. Moreover, conventional 2 or 3-color systems used with conventional ELISA or ELISPOT methods cannot separately resolve individual spectral features of each colored detection reagent. In conventional multicolor assays, development of colors typically is made sequentially, one after another, each followed by washing steps. This procedure is time consuming and because of its complexity is difficult to carry out. Additionally, substrates are not typically fully transparent, so a second substrate may mask an underlying substrate. Moreover, as more substrates are used, the background becomes darker and less controlled. The above problems with conventional ELISA methods therefore make assays of multiple cellular products difficult or impossible.
There is a great need for better assays to measure secreted soluble cell products. Specifically, there is a need for devices and methods with greater capability to detect cytokines from individual cells in a mixture of heterogeneous cells, and for methods that can detect multiple different secreted cytokine cell products within mixtures of such products, and for methods that have increased reliability and reproducibility.